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Identification of Protected Deoxyribonucleotides by Field Desorption Mass Spectrometry in Fractions from High Performance Liquid Chromatography* H a n s Mart in Schiebel*a and Hans-Rolf Schultenb

a Institute of Organic Chemistry, Technical University Braunschweig, Schleinitzstraße 2, D-3300 Braunschweig

b Institute of Physical Chemistry, University Bonn, Wegeierstraße 12, D-5300 Bonn

Z. Naturforsch. 36b, 967-973 (1981); received April 6, 1981

Protected Deoxyribonucleotides, Field Desorption, HPLC

Protected deoxyribonucleotides were identified by field desorption mass spectrometry in eluate fractions from high performance liquid chromatography. The production of abundant cationized molecules and the formation of structural significant fragments by thermally/field induced processes allowed a direct and unambiguous identification of the synthesized products. In addition, indications can be obtained on both organic and inorganic impurities.

Introduction Fully and part ial ly protected mono- and oligo-

deoxyribonucleotides have been used as starting materials in sequence specific synthesis of poly-nucleotides [2, 3]. These substances are of consider-able interest as seen f rom recent investigations in genetic engineering [4-6].

The first systematic studies by mass spectrometry of the synthesis control of protected mono- and oligodeoxyribonucleotides were made by Arm-bruster and Wiebers [7]. However, the lability of protected nucleotide di- and triesters upon thermal and electronic excitation allowed only limited in-format ion t o be obtained using electron impact (EI) mass spectrometry (MS). I n most cases the spectra revealed no direct indication of the molecular ions of the invest igated nucleotides. Thus, using conven-tional E I MS identification is only possible in-directly b y analysis of degradation products. However, recent results by californium-252 plasma desorption [8] and the application of field desorption (FD) presented in this s tudy demonstrate the analytical capaci ty of al ternative mass spectro-m e t r y ionization techniques in the field of nucleic acid research.

I t has been demonstrated tha t F D MS is a suitable mass spectrometric method for molecular weight de terminat ion and, to some extent , structural

+ Field Desorption Mass Spectrometry of Nucleic Acids VII. For part VI see ref. [1].

elucidation of free nucleosides and mononucleotides [9] a n d of free dinucleoside phosphates [10-12]. I n general, it appears t h a t F D MS is a powerful method for t h e detection, identification and quantification of highly polar compounds in biochemical, medical and environmental analysis [13].

I n the following we report the first F D MS results of ful ly and part ial ly protected building blocks for polynucleotide synthesis which allow both molecular weight determinat ion and elucidation of s tructural information. The analytical aim of this investigation was identification and pur i ty control of deoxyribo-nucleotides following high performance liquid chro-matography (HPLC). This initial systematic F D MS s tudy is of substances (1-9) of relatively low polarity b u t particularly high chemical and thermal lability.

B R o - ^ A j

0 0 - C H 2 - C H 2 - C S N

1 - 8

1 B = thymine 2 = N-benzoylcytosine 3 = N-benzoyladenine 4 = N-isobutyrylguanine 5 = thymine 6 = N-anisoylcytosine 7 = N-benzoyladenine 8 = N-isobutyrylguanine

R = £>,.p'-dimethoxy-trityl

R = H

Results and Discussion

A. Fully protected deoxyribomononucleotides

Molecular weight determination: Since all sub-stances 1-4 gave intense molecular ions, [M-(-H]+

and [M-fNa]+ , respectively, unambiguous molecular weight determination was achieved. These intense molecular ions can be obtained under soft ionization conditions. For instance, as shown in Fig. 1 a under these conditions compound 1 yields the [M-fH]+

ion at m\z 788 as base peak of the spectrum. The detection of these spectra is somewhat critical as the fully protected nucleotides desorb over a small temperature range and within a short period of time. Thus, for the spectrum in Fig. 1 a the section of the total desorption process at the best anode temperature [13] could only be recorded in a single magnetic scan. When a number of repetitive scans were accumulated by the data system and a more extended range of the emitter heating current was applied the relative abundance of the ion [M-)-H]+

drops drastically. Formation of [M-fNa]+ a t m\z 810 by attachment of sodium ions to the original molecule competes with the formation of [M-f H]+ at m\z 788 (Fig. lb) . This cationization [13] of the molecule is the favoured process in the case of compound 2 (m/z 899), 3 (m\z 923) and 4 (m/z 905). I t is noteworthy that in all experiments the im-purities of alkali salts present in the authentic samples were sufficient for cationization.

Structural information: As shown in Fig. 1 a under soft FD conditions (low emitter heating current) structural information is somewhat limited as only the molecular ion and one fragment at m\z 303 can be observed. The chemical lability of the bond between the dimethoxytrityl group and the deoxy-ribose moiety is reflected in its pronounced cleavage under F D conditions. The high relative abundance of the dimethoxytrityl fragment at m\z 303 is a characteristic feature in the FD spectra of all dimethoxytrityl protected nucleotides. Slight in-crease of the emitter temperature gives a signifi-cantly different spectrum. The signal for the protecting dimethoxytrityl group becomes the base peak in the spectrum. Simultaneously some addi-tional ions can be detected (Fig. 1 b). In general, the most important degradation reaction is the forma-tion of the protected [1-4) or unprotected (1) base by cleavage of the N-glycosidic bond (e.g. m\z 126, Fig. lb ) . Proton attack on the 3'-phosphoric acid

ester bond yields p-chlorophenyl-ß-cyanoethyl-phosphoric acid. Subsequent protonation of this neutral degradation product on the emitter surface produces the signal at m\z 261 showing the charac-teristic isotopic pattern of a compound containing one chlorine atom. This fragment can be observed for the pyrimidine nucleotides 1 and 2 but is missing in the spectra of the purine nucleotides 3 and 4 (see section B). A signal at m\z 224 can be assigned to an ion B-f-99. Its formation occurs presumably by initial hydrolytic cleavage of the dimethoxytrityl group and subsequent elimination of the phosphate moiety. I t is detected with low relative abundance (6%) in the spectrum of compound 1 (Fig. 1 b). The analogous ions at m\z 313, 337, and 319 in the spectra of the compounds 2, 3, and 4, respectively, were not observed. However, this fragment is im-portant in the spectra of 5 and 6 (see section B). By analogy with general decomposition reactions in E I MS fragmentation mechanisms could be formu-lated leading to the ion signals at m\z 288, 334, and 748, respectively. But under FD conditions these degradation reactions are not very probable. In addition, these fragmentations should be charac-teristic for the FD spectra of the nucleotides in-vestigated here. This is not the case. Therefore the intense signal at m\z 288 and the signals at mjz 334 and mjz 748 in the spectrum of compound 1 (Fig. 1 b) may represent the molecular ions of impurities and/or their degradation products produced by pyrolytic reactions on the emitter surface.

B. Partially protected, functionalized deoxyribomononucleotides

Molecular weight determination: The slightly higher polarity of the functionalized mononucleo-tides 5-8 with an unprotected 5'-OH-group favours protonation and cationization of the molecules and enables unequivocal molecular weight determina-tion by the ions [M-fH]+ and [M-fNa]+, respec-tively (Fig. 2).

At emitter heating currents sufficient for distinct fragmentation the relative abundance of the pro-tonated molecules 5-8 was between 16-35% and the cationized molecules generated by [Na]+ attachment were between 3 and 17%. I t is noteworthy tha t in all cases for 5-8 both [M-f H]+ and [M+Na]+ ions could be detected and utilized for confirmation of the molecular weight. The [M]+ ion was not ob-

1 a

1 b

100-]

8 0 -

60

40

20-|

0

v I T H a C O - ^ - C - ^ o — , 1 ,125

Mol. wt. = 787.206

50 100 <

* 100-1

80-

6 0 -

40-

2 0 -

400 600

100-i

8 0 -

60

40-

20-

0 3 -Q < "3

* 100-j

8 0 -

60-

40-

20-

53

50 100

400

CH2

CH2 1

c III N

-i 1 r i-150 200 250

—i 1 1 1 r 300 350

[M+H]+

788

650 700 750 800 850

m/z

288 I

126 I

224 I

261 I

I ' ' 1

150

,303

334 I

200 250 300

[m+H]+

788

350

748 I

' I 1

600

[M+Na]*

810 I

650 700 750 800 850

m/z

400

900

""I 400

—I 900

2 a

Mol.wt. = 580.124

222

244 261

400

900

2 b 100

80-

6 0 -

40

20H

0

58

50

100-,

< 80 H

6 0 -

40-

20-

0

98

100

443

128

150

450 500

200

221

261

244

250

[m+H]+

5 8 1 [M+Na] +

603

302

300

550

m/z

600 650

350 400

900

served. The relative abundance of t he molecular ions are strictly dependent on the conditions applied in the same way as those for the dimethoxytr i ty l compounds 1-4. For example for 8 the relative abundance of the ion [M+H]+ a t m/z 581 decreases f rom 100% to 10% with increasing emit ter tem-perature (Fig. 2).

Structural information: I n general, t he same frag-mentat ion behaviour as for compounds 1 -4 can be observed a t emitter temperatures with slightly increased heating cm-rents above the best anode temperature. Surprisingly, the base ion in the spectra of the pyrimidine nucleotides 5 and 6 is t he fragment of the phosphate moiety a t mjz 261. I n contrast to this result, the base peak in the spectra of the purine nucleotides 7 and 8 is the protected nucleobase a t mjz 239 and mjz 221 (Fig. 2 b), respectively. On the basis of recent in-vestigations of purine-pyrimidine-polynucleotides by pyrolysis F D MS the reversed behaviour was expected because of the somewhat higher volati l i ty and ionization efficiency of the pyrimidines thymine and N-anisoylcytosine. Presumably, in this case the enhanced lability of the N-glycosidic bond of base protected purine nucleotides against proton-induced cleavage is the reason for this behaviour. The preferred rupture of the N-glycosidic bond of the purine nucleotides 7 and 8 prevents the format ion of a f ragment B - j-99 which can be detected in the F D spectra of the pyrimidine nucleotides 5 and 6. This fragment is complementary to t h a t observed a t mjz 261 for the protected phosphate moiety. 2 ' , 3 ' - Dideo xy - 2 ' , 3 ' - d e h y d r o - p yr i mi d ine -riboside may be formulated as a possible structure. The signals a t mjz 301 and mjz 302 in the spectrum of compound 8 (Fig. 2 b) may represent the [M]+ and the [M-)-H]+ ion of a degradation product of the nominal mass B-f-81 which was first observed as a key ion in the pyrolysis E I [14] an F D [15] spectra of DNA and identified by high resolution. The structure of this f ragment was suggested to be a methylfuran derivative of t he appropriate nucleo-side [14]. The relative abundance of the B + 8 1 ion amounts to 10% in the spectrum of compound 6 bu t is not detectable in the spectra of 5 and 7. The relatively intensive signals a t mjz 244 and 443 in the spectrum of 8 (Fig. 2) are assumed to be due to an ion [ 2 B + H ] + (m/z 443) and to an ion formed by water elimination f rom the pro tonated phosphoric

acid moiety (m/z 244). However, t he expected iso-topic pa t te rn is missing for these ions. Only one signal is detected by the da ta system in both cases. For the present the structural assignment for the ions a t mjz 443 and mjz 244 has to remain more speculative. The ion a t mjz 128 indicates the p-chlorophenyl moiety of the phosphate triester and the f ragment a t m/z 98 corresponds by its nominal mass to a degradation product of deoxyribose found in the high resolution F D spectra of DNA [15]. I t s s tructure had been identified by collisional activa-t ion mass spectrometry as a mixture of a-angelica lactone and furfuryl alcohol [16].

C. Partially protected, functionalized deoxyribo-dinucleoside-diphosphates

Molecular weight determination: I n Fig. 3 the F D spectrum of compound 9 is displayed as the first example of a dinucleotide building block. Also in this case the very clear and simple spectrum enables reliable molecular weight determination from the molecular ions of high relative abundance a t mjz 1102 for [M+H]+ and a t mjz 1124 for [M+Na]+. I n addition, this determination is supported by the following doubly charged molecular ions: [M]++

a t mjz 550.5, [ M + 2 H]++ a t mjz 551.5 and [ M + H + N a ] + + a t m/z 562.5. This formation of doubly charged ions could not be observed for the mononucleotides 1-8.

Structural information: The base peak of the spectrum is formed a t m/z 314 by fission of t he 3'-ester bond of the 5'-terminal nucleoside and release of a protonated 2 , ,3 /-dideoxy-2',3'-dehydro-riboside. As mentioned above, this f ragment was fond to be characteristic in the spectra of t he pyrimidine-nucleotides 5 and 6. Both nucleobases can be observed as protonated species a t m/z 216 and mjz 240, respectively. One of the most impor tant f ragmentat ions is obtained by rupture of the 5'-ester bond and proton a t tachment to the phosphate moiety of the 5'-terminal nucleoside yielding the complementary fragments 522 and 581. We assume t h a t both f ragment ions are formed via protonat ion of the neutral species produced on the emit ter surface during the F D process. The neutral species of mass 521 represents the deoxycytidine-3'-phos-pha te residue of the original molecule where as the neutral species of mass 580 corresponds to 5'-deoxy-4',5'-dehydro-deoxyadenosine-3'-phosphate formed

100-

8 0 -

50-

40

20

100-,

® 8 0 -

® 60-

40-

20-

u

314

H 0 T 0 ^ 2 U H - J - i - Q

? r \ x j 0 = p — 0

216

240

200 r/fr —i-7/i—r—

250 300

314

238

Mo l .wt . = 1101.178

O H Q k c i

551.5 581 522 | I

-i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—i—r—i—i—r—i"—i—r—i—i 350 400 450 500 550 600

[m+H] 1102

• [M • Nq]+

1124

-i—i—i—i—i—i—i—i—i—i— 600 650 700 750

m /z

—i—i—i—i—i—T"~i—O—i—i—r-1050 1100 1150

—I 1200

Fig. 3. FD mass spectrum of compound 9. Emitter heating current 20 mA. Average of 8 spectra by the data system.

af ter cleavage of the 5'-ester bond of the dinucle-otide 9. Finally, a relatively low abundant ion a t m/z 128 indicates the p-chlorophenyl moiety of compound 9.

This investigation demonstrates t h a t F D MS is not only a suitable tool for molecular weight deter-minat ion of protected mono- and dinucleotides, bu t allows significant s tructural details to be elucidated. I n view of the results obtained with protected dinucleotides this method should also be useful for the analysis of larger oligomers.

Experimental The F D measurements were performed on a

type 731 Varian MAT double-focusing mass spectro-

meter equipped with a combined commercial E I / F D ion source. The F D emitters used were 10 jum tungs ten wires act ivated a t high temperature. The length of the carbon microneedles was 40 ^m on average. The molecular ion of acetone (m/z 58) was used for ad jus tmen t and calibration of the emitter. All spectra were produced using direct heating of the emit ter by a heating current. The sample was t ransferred to the emit ter by the modified syringe technique [13] and in general 1 to 3 jug of sample mater ia l were deposited on the center of the front side of the emit ter . The applied potentials were -f 8 kV for the field anode and — 4 kV for the opposing cathode plate. The F D ion currents were recorded electrically in combination with the Varian SS 200 da ta system. All spectra were acquired a t a mass resolution of about 2000 (10% valley definition). The analysis t ime for the identification

of a nucleotide in fractions of HPLC and the analysis of its pur i ty was 30 min. This includes sample transfer , emit ter adjus tment , field desorp-tion, d a t a acquisition and processing (interpreta-tion). Exper imenta l details for the HPLC separation are given in ref. [1].

This work was supported by the Deutsche For-schungsgemeinschaft (Schi 168/4), (Schu 416/3) and the Ministerium fü r Wissenschaft und Forschung des Landes Nordrhein-Westfalen. The generous gift of compound 1 - 9 by Prof. Dr. H . Seliger, University of Ulm, FRG, is gratefully acknowledged.

[1] H. Seliger, T. C. Bach, H.-H. Görtz, E. Happ, M. Holupirek, H. M. Schiebel, and H.-R. Schulten, Anal. Biochem., in preparation.

[2] C. B. Reese, Tetrahedron 84, 3143 (1978). [3] Proceedings of the International Symposium on

Chemical Synthesis of Nucleic Acids, held in Egestorf, GFR, on 5th-8th May 1980. Nucleic Acids Symposium Series No. 7, Köster, H. (ed.), 1980.

[4] R. Crea, A. Kraszewski, T. Hirose, and K. Itakura Proc. Natl. Acad. Sei. USA 75, 5765 (1978).

[5] H. M. Hsiung, R. Brousseau, J. Michniewicz, and S. A. Narang, Nucl. Acids Res. 6, 1371 (1979).

[6] H. Seliger, T. C. Bach, E. Happ, M. Holupirek, and E. H. Teufel, Hoppe-Seyler's Z. Physiol. Chem. 360, 1044 (1979) and literature cited.

[7] M. A. Armbruster and J. L. Wiebers, Anal. Biochem. 83, 570 (1977).

[8] C. J. McNeal, S. A. Narang, R. D. Macfarlane,

H. M. Hsiung, and R. Brousseau, Proc. Natl. Acad. Sei. USA 77, 735 (1980).

[9] H.-R. Schulten and H. D. Beckey, Org. Mass Spectrom. 7, 861 (1973).

[10] H.-R. Schulten and H. M. Schiebel, Z. Anal. Chem. 280, 139 (1976).

[11] H.-R. Schulten and H. M. Schiebel, Nucl. Acids Res. 3, 2027 (1976).

[12] H. Budzikiewicz and M. Linscheid, Biomed. Mass Spectrom. 4, 103 (1977).

[13] H.-R. Schulten, Int. J. Mass Spectrom. Ion Phys. 32, 97 (1979) and literature cited.

[14] G. A. Charnock and J. L. Loo, Anal. Biochem. 37, 81 (1970).

[15] H.-R. Schulten, H. D. Beckey, A. J. H. Boer-boom, and H. L. C. Meuzelaar, Anal. Chem. 45, 2358 (1973).

[16] K. Levsen and H.-R. Schulten, Biomed. Mass Spectrom. 3, 137 (1976).